The lifetime and abundance of RNA is regulated by maintaining a balance between RNA transcription and RNA degradation. While several RNA degradation pathways have been discovered, their components, precise catalytic activities, and cellular function remain a continued focus of study. One of two principle RNA decay pathways in eukaryotes involves the RNA exosome, a multi-subunit protein complex that catalyzes 3'to 5'RNA decay. The RNA exosome is conserved throughout eukaryotic evolution and is thought to exist in at least two forms, a cytoplasmic exosome composed of nine or ten distinct protein subunits with an apparent mass of 300-400 kDa, and a nuclear exosome composed of ten or eleven distinct protein subunits with an apparent mass of 400-500 kDa. In budding yeast, ten of the eleven genes are essential for growth, suggesting critical roles for each in cellular function. The identification of individual exosome subunits led to the hypothesis that the exosome was composed of up to eleven distinct 3'to 5'exoribonucleases. However, recent studies utilizing reconstituted and affinity-purified cellular exosome complexes indicate that only a few of the subunits encode polypeptides with exoribonuclease activity, suggesting that most exosome subunits are conserved for their non-catalytic functions. Consistent with this, exosome subunits are known to associate with a variety of protein partners that facilitate or direct RNA degradation. While recent efforts have revealed many interesting aspects of eukaryotic exosome structure and function, many questions remain with respect to individual and collective functions for exosome subunits in RNA decay. In this proposal, we will utilize in vitro reconstituted human and yeast exosome complexes in combination with genetic and biochemical assays to analyze its assembly, catalytic activity, and structural integrity by conducting research with these aims: 1) characterize and reconstitute subunits and complexes from human and yeast exosomes;2) determine the biochemical basis for activities ascribed to individual, sub-complexes, and intact exosome complexes from yeast and human and determine the physiological importance of the observed activities and structures through in vivo complementation and analysis in the budding yeast Saccharomyces cerevisiae;3) determine the structural and biophysical basis for exosome subunits and complexes from yeast and human. RNA exosomes contribute to cellular RNA homeostasis through 3'to 5'decay, thus balancing RNA transcription with RNA degradation. Exosomes are also involved in maintaining RNA integrity via several quality control pathways which serve to target aberrant RNA for destruction. Together, these pathways regulate the lifetime of a particular RNA and protect the cell from deleterious RNA that could lead to cellular pathology. Defects in these processes are associated with several human diseases including cancer, inflammation, and neurodegenerative disorders.